Recent studies provided evidence that, in addition to DNA damage, ionizing radiation acts upon cellular membranes to initiate apoptotic death in some cells. Genetic, biochemical and cell biologic data generated during the last funding period established a critical role for acid sphingomyelinase (ASMase)-mediated ceramide generation in signaling radiation-induced apoptosis in select cells such as oocytes and endothelium. Further, ASMase-mediated apoptosis was crucial for tissue radiation responses in the ovary, GI tract, lung and tumor models. The hypothesis of the current proposal is that a transmembrane signaling mechanism, recently discovered by our laboratory, involving ceramidedriven re-organization of membrane rafts into large signaling platforms, affords a mechanism by which radiation induces endothelial apoptosis. Furthermore, the proposed research will test whether genetic up-regulation of this process serves to sensitize tumors to radiation. The proposal contains 3 specific aims. Aim 1 surveys the extent to which epithelial and non-epithelial tumor xenografts undergo endothelial cell apoptosis in vivo after single dose radiation. The role of microvascular dysfunction in GI and tumor responses will be determined in multiple mouse strains, as we have now bred the asmase knockout onto 3 separate backgrounds. Further, the genetics of radiationinduced endothelial apoptosis will be investigated by transplanting tumors into mice lacking genes involved in DNA damage repair (i.e. p53, ATM, SCID) or apoptosis (i.e. Bax, ASMase, BID). Aim 2 addresses the pro-apoptotic mechanism of ceramide-mediated apoptosis at the cellular and sub-cellular level. Initial single cell oocyte studies using particle microbeam irradiation will establish whether the SM Pathway induces apoptosis independent of or in concert with DNA damage. Subsequent studies will address whether ceramide-driven re-organization of microscopic membrane rafts into large signaling platforms is the mechanism of radiation-induced endothelial cell death. Aim 3 uses a gene therapy approach to modulate ASMase-mediated endothelial cell platform formation and apoptosis within tumors using retroviral and lentiviral vectors generated to overexpress asmase. In particular, bone-marrow-derived endothelial progenitors will be targeted to manipulate apoptosis within developing tumor microvasculature and influence tumor growth rate and radiation response. If results indicate that overexpressing ASMase effectively radiosensitizes tumor microvasculature, applicability to human tumor radiotherapy will be examined directly using human versions of lentiviral asmase vectors.